Expression in insect cells of genes with overlapping open reading frames, methods and compositions therefor

ABSTRACT

The present teachings disclose nucleic acid cassettes for expressing in an insect cell a plurality of polypeptides encoded by a gene comprising overlapping open reading frames (ORFs). A cassette comprises, in 5′ to 3′ order, a) a first insect cell-operable promoter, b) a 5′ portion of a gene comprising a first ORF of the gene, c) an intron comprising a second insect cell-operable promoter, and d) a 3′ portion of the gene comprising at least one additional ORF. Vectors and insect cells comprising the cassettes are also disclosed, as well as methods for production of recombinant adeno-associated virus in insect cells using the cassettes.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application claims priority to U.S. Provisional Patent Application60/839,761 filed Aug. 24, 2006, which is incorporated herein byreference in its entirety.

INTRODUCTION

Genes comprising overlapping reading frames (ORFs) are known to existwithin mammalian genomes. In some cases, such genes comprise an intronwhich has a promoter which supports transcription of an ORF (See, e.g.,Reisman, D., et al., Proc. Nat'l. Acad. Sci. USA 85: 5146-5150, 1988;Bennett, V. D., et al., J. Biol. Chem. 265: 2223-2230, 1990). However,no insect gene, either naturally occurring or artificial, has beenreported which encodes multiple ORFs and comprises a promoter within anintron.

Parvoviridae comprise a family of single-stranded DNA animal viruses.The family Parvoviridae is divided between two subfamilies: theParvovirinae, which infect vertebrates, and the Densovirinae, whichinfect insects. The subfamily Parvovirinae (members of which herein arereferred to as the parvoviruses) includes the genus Dependovirus, themembers of which are unique in that, under most conditions, theseviruses require coinfection with a helper virus such as adenovirus orherpes virus for productive infection in cell culture. The genusDependovirus includes adeno-associated virus (AAV), which normallyinfects humans and primates (e.g., serotypes 1, 2, 3, 3A, 3B, 4, 5, 6,7, 8, 9, 10, & 11), and related viruses that infect other warm-bloodedanimals (e.g., bovine, canine, equine, and ovine adeno-associatedviruses). The parvoviruses and other members of the Parvoviridae familyare generally described in Kenneth I. Berns, “Parvoviridae: The Virusesand Their Replication,” Chapter 69 in FIELDS VIROLOGY (3d Ed. 1996) andrecent review article by Choi et al., Curr Gene Ther., June;5(3):299-310 (2005).

The AAV genome is a linear, single-stranded DNA molecule that is lessthan about 5,000 nucleotides (nt) in length. Inverted terminal repeats(ITRs) flank the unique coding nucleotide sequences for thenon-structural replication (Rep) proteins and the structural (VP)proteins. The ITRs are self-complementary and are organized such that anenergetically stable intramolecular duplex T-shaped hairpin can beformed. These hairpin structures function as an origin for viral DNAreplication. The Rep genes encode the Rep proteins, Rep78, Rep68, Rep52,and Rep40. Rep78 and its splice variant Rep68 are translated from mRNAsthat are transcribed from the p5 promoter. Rep52 and its splice variantRep40 are translated from mRNAs that are transcribed from the p19promoter. The Cap genes encode the VP proteins, VP1, VP2, and VP3. Theseproteins form the capsid, and are synthesized from two spliced mRNAsarising from transcription of the Cap gene from the p40 promoter. Onemessage is translated into VP1, while another transcript encodes VP2 andVP3. The naturally occurring initiation codon for VP2 is an ACG, whichis poorly utilized, resulting in ribosome scanning through to the VP3initiation codon (AUG). The alternate usage of two splice acceptor sitesfor the VP1 transcript and the poor utilization of ACG initiation codonfor VP2 are believed responsible for the stoichiometry of VP1, VP2, andVP3 in AAV2-infected mammalian cells and mirrors the protein ratio inthe capsids, 1:1:10. Urabe, M. et al., Hum. Gene Ther. 13: 1935-1943,2002.

An ITR of an AAV is known to function as an origin of replication, i.e.,a site having a “cis” role in replication, and serves as a recognitionsite for trans acting replication proteins (e.g., Rep 78 or Rep68) whichrecognize the palindrome and specific sequences internal to thepalindrome. One exception to the symmetry of the ITR sequence is the “D”region of the ITR. It is unique (not having a complement within oneITR). Nicking of single-stranded DNA occurs at the junction between theA and D regions. It is the region where new DNA synthesis initiates. TheD region normally sits to one side of the palindrome and providesdirectionality to the nucleic acid replication step. An AAV replicatingin a mammalian cell typically has two ITR sequences.

In mammalian cells, the four Rep proteins of AAV are encoded by multipletranscripts from a single reading frame. Promoters at map positions 5and 19 regulate transcription of the Rep ORF. Rep78 and 68 are expressedfrom the p5 promoter and differ from each other by a 3′-splice. Rep68 isa carboxy-truncated version of Rep78, although Rep68 contains 7 uniqueresidues as a result of a frame shift occurring in the splice acceptorsite. The Rep52 and Rep40 transcripts are expressed by the p19 promoterand are in-frame with the larger Rep polypeptides. The smaller Reppolypeptides differ from each other in the same manner as Rep78 andRep68, i.e., by a splicing event. The functional domains of Rep are:Amino terminus-DNA binding-DNA nicking-ATPase-Helicase-nuclearlocalization signal-modified zinc finger-COOH. The functions of DNAbinding and DNA nicking are present only in the Rep proteins transcribedfrom p5.

AAV replicates via a duplex DNA intermediate that is one continuousmolecule: both strands are covalently attached through the ITR. The p5Rep proteins are able to recognize a motif within the ITR, nick onestrand of the duplex and become covalently attached through thetyrosinyl-thymidine phosphodiester linkage at the 5′-side of the nick.The helicase activity of Rep is believed responsible for unwinding thenewly created 5′-end, and a cellular polymerase complex extends therecessed 3′-end to generate a duplex, blunt-ended replicationintermediate. The smaller Rep proteins retain the ATP-dependent, DNAhelicase activity and are involved in encapsidation of thesingle-stranded virion genomes. Rep52 and Rep40 associate with preformedcapsids and, presumably, unwind the duplex replication intermediates.

In recent years, AAV has emerged as a preferred viral vector for genetherapy due to its ability to infect efficiently both nondividing anddividing cells, integrate into a single chromosomal site in the humangenome, and pose relatively low pathogenic risk to humans. In view ofthese advantages, recombinant adeno-associated virus (rAAV) presently isbeing used in gene therapy clinical trials for hemophilia B, malignantmelanoma, cystic fibrosis, and other diseases.

AAV sequences employed for the production of AAV in insect cells can bederived from the genome of any AAV serotype. Generally, the AAVserotypes have genomic sequences of significant sequence identity at theamino acid and the nucleic acid levels, provide a virtually identicalset of genetic functions, produce virions which are essentiallyphysically and functionally equivalent, and replicate and assemble bypractically identical mechanisms. For the genomic sequence of AAVserotypes and a discussion of the genomic similarities see, for example,GenBank Accession number U89790; GenBank Accession number J01901;GenBank Accession number AF043303; GenBank Accession number AF085716;Chiorini et al., J. Vir. 71: 6823-33 (1997); Srivastava et al., J. Vir.45:555-64 (1983); Chiorini et al., J. Vir. 73:1309-1319 (1999); Rutledgeet al., J. Vir. 72:309-319 (1998); and Wu et al., J. Vir. 74: 8635-47(2000).

The difficulties involved in scaling-up rAAV production using currentmammalian cell production systems can be significant, if not entirelyprohibitive. For example, for certain clinical studies more than 10¹⁵particles of rAAV may be required. To produce this number of rAAVparticles in a mammalian cell line such as human 293 cells, transfectionand culture of approximately 10¹¹ cells, the equivalent of 5,000 175-cm²flasks of cells, would be required. There also is the possibility that avector destined for clinical use produced in a mammalian cell culturewill be contaminated with undesirable, perhaps pathogenic, materialpresent in a mammalian cell.

U.S. Pat. No. 6,723,551 B2 to Kotin et al., and Urabe et al., J. Virol.80: 1874-1885, 2006 disclose methods of producing rAAV vectors in insectcells. In these disclosures, baculovirus vectors were constructed thatinclude nucleic acids that encode Rep78/68 and Rep52/40 in a palindromichead-to-tail arrangement, each of Rep78/68 and Rep 52/40 genes under thecontrol of an independent promoter (FIG. 4 herein). These vectorsinclude inverted terminal repeats (ITRs), transgene-encoding sequencesand AAV capsid genes. While high titer rAAV was initially produced,there was no evidence that the method would be adaptable to large-scaleproduction of rAAV. In addition, several research groups have reportedthat using the specific design of VP1 expression as described in U.S.Pat. No. 6,723,551 B2 produces less infectious AAV vectors as comparedwith their 293 cell produced counterpart. Merten et al., Gene Ther., 12:S51-S61, 2005; Kohlbrenner et al., Mol. Ther., 12: 1217-1225, 2005.

The U.S. Pat. No. 6,723,551 B2 patent also asserts that Rep mRNAsplicing in insect cells does not mimic the process in mammalian cells.In particular, this reference teaches modifying a Rep68 or Rep40 codingnucleotide sequence so as to be devoid of an intron. A nucleic acidsequence to be translated in an insect cell by these methods includesonly the coding sequence. Therefore, the coding sequence of any viralmRNA transcribed in this patent avoids the splicing out (removal) of anintron before translation. However, in order to express both Rep78 andRep52 in insect cells, the methods set forth in U.S. Pat. No. 6,723,551B2 use two separate Rep sequences and two expression cassettes, one forRep78 and the other for Rep52.

In mammalian-cell produced AAV, the best yield of “full” virions (i.e.,viral particles incorporating an AAV genome), that are fully functionaland can, for example, target the nucleus, is obtained when all three VPproteins are expressed, and they are at a stoichiometry approaching1:1:10 (VP1:VP2:VP3). The regulatory mechanisms that allow thiscontrolled level of expression include the production of two mRNAs, onefor VP1, the other for VP2 and VP3, produced by differential splicing.

The splicing event required to produce a 1:1:10 stoichiometry ofVP1:VP2:VP3 is not properly reproduced in the insect cells when originalAAV Cap coding sequence is used. The majority of proteins expressed fromthe Cap coding sequence is VP1, which is initiated from the first AUGcodon of the Cap coding sequence. In order to mimic the 1:1:10stoichiometry of VP1:VP2:VP3, the methods set forth in U.S. Pat. No.6,723,551 B2 mutates the AUG codon of VP1 into an ACG to decrease theexpression level of VP1.

Although AAV intron does not properly function in insect cells, it hasbeen reported that the immediate-early IE-1 gene of Autographcalifornica nuclear polyhedrosis virus comprises a functional intronthat can be spliced in insect cells (Chisholm and Henner, J. Virol., 62:3193-3200, 1988). However, this reference does not suggest incorporatinga promoter within the intron for expressing an operably linked openreading frame.

Various attempts have been applied to enhance the expression of VP1 ofAAV, such as using chimeric VP1 protein or “riboswitch” mechanisms. See,e.g., Urabe et al., J. Virol., 80: 1874-1885, 2006 and Kohlbrenner etal., Mol. Ther., 12: 1217-1225, 2005, and U.S. patent application2006/0166363. Furthermore, the recombinant baculovirus as described inU.S. Pat. No. 6,723,551 B2 harboring large homologous repeats of Rep78and Rep52 has been reported unstable. Kohlbrenner et al., Mol. Ther.,12: 1217-1225, 2005. Although Kolhlbrenner et al. reported baculovirusesthat are more stable and AAV vector more infectious compared to those ofthe U.S. Pat. No. 6,723,551 B2 patent, their techniques requireseparation of the coding sequences for Rep78 and Rep52 into twobaculoviruses, and an additional baculovirus to supply VP1 protein.

Cao, L. et al. (J. Virology 74: 11456-11463, 2000) described a methodfor producing a high-titer, wild-type free recombinant adeno-associatedvirus vector using intron-containing helper plasmids. These methodsinvolve insertion of a human beta-globin intron into the AAV genome.However, these methods use human cells as host, and, in addition, theintroduced intron does not include a promoter that can directtranscription in insect cells.

Therefore, there remains a need for improved methods and nucleic acidsfor expressing genes with overlapping open reading frames in insectcells, as well as methods, nucleic acids and cells for producinginfectious parvoviral vectors.

SUMMARY

In view of the need for methods of expressing genes comprisingoverlapping open reading frames (ORFs) in insect cells, the presentinventor has developed nucleic acids, cells, cell cultures, and methodsfor expressing genes comprising ORFs.

In some aspects, the present teachings disclose nucleic acid cassettes.These cassettes can be used for expressing, in an insect cell, aplurality of polypeptides encoded by a gene comprising overlapping openreading frames (ORFs). A cassette of these aspects comprises, in 5′ to3′ order: a) a first insect cell-operable promoter; b) a 5′ portion ofthe gene comprising a first ORF of the gene; c) an intron comprising asecond insect cell-operable promoter; and d) a 3′ portion of the genecomprising at least one additional ORF of the gene, wherein the firstinsect cell-operable promoter is operably linked to the first ORF, thefirst ORF comprises a first translation initiation codon; the secondinsect cell-operable promoter is operably linked to the at least oneadditional ORF, and the at least one additional ORF comprises at leastone additional translation initiation codon, and wherein at least one ofa), b), c) and d) can be heterologous to at least one other of a), b),c), and d). In various configurations, a cassette can further comprisee) a polyadenylation signal situated 3′ to d). In related aspects, atleast one of a), b), c), d) and e) can be heterologous to at least oneother of a), b), c), d) and e). In various configurations, the presentteachings include vectors which comprise a nucleic acid cassette. Avector can be any type of vector known to skilled artisans, for example,a plasmid, a virus, a viral nucleic acid, or a combination thereof. Inaddition, in various configurations, a vector or a nucleic acid cassettecan be a single- or double stranded DNA, and a virus can be abacteriophage or a baculovirus.

In various configurations, a nucleic acid cassette of these aspects caninclude insect cell-operable promoters (i.e., promoters which supporttranscription in an insect cell). A cassette can comprise any insectcell-operable promoter known to skilled artisans. In some aspects, afirst insect cell-operable promoter of a cassette can be a p10 promoteror a polh promoter and, independently, a second insect cell-operablepromoter can be a p10 promoter or a polh promoter.

In various aspects of the present teachings, a gene comprisingoverlapping ORFs can be a gene of a virus, such as a virus that infectsmammalian cells. In some aspects, the virus can be an adeno-associatedvirus (AAV). In some configurations, a nucleic acid cassette cancomprise a first insect cell-operable promoter, a 5′ portion of a Repgene of an AAV or a 5′ portion of a Cap gene of an AAV, and an introncomprising a second insect cell-operable promoter. In some arrangements,a nucleic acid comprising a Rep gene can include a Rep 78/68 ORF as afirst Rep ORF and a Rep 52/40 ORF as a second or additional ORF, asdescribed infra. Furthermore, in some configurations, the first promotercan be a p10 promoter and a second promoter can be a polh promoter. Inother configurations, a nucleic acid cassette can include a Cap gene inwhich a first ORF can be a VP1 ORF and a second or additional ORF can bea VP2/VP3 ORF, as described infra. In some configurations, both thefirst and the second promoters of a nucleic acid cassette can be a polhpromoter.

In yet other configurations, a single nucleic acid can comprise two ormore cassettes, each cassette comprising a different gene comprisingmultiple ORFs. A nucleic acid can comprise cassettes arranged in atandem, i.e., with the same polarity, or in an anti-sense orientation.Hence, a single nucleic acid in some configurations can comprise a firstinsect cell-operable promoter, a 5′ portion of a Rep gene of an AAV, afirst intron comprising a second promoter, a 3′ portion of the Rep gene,a third insect cell-operable promoter, a 5′ portion of a Cap gene, asecond intron comprising a fourth insect cell-operable promoter, and a3′ portion of a Cap gene. In addition, in some aspects, apolyadenylation signal can be positioned 3′ to each 3′ portion.

The present teachings also include an insect cell comprising one or morenucleic acid cassettes which can be used to express in an insect cell aplurality of polypeptides encoded by a gene comprising multiple ORFs. Aninsect cell of these teachings can be an insect cell in vitro, such asan insect cell from a cultured cell line such as a BTI-Tn-5B1-4 fromTrichoplusia ni (High-five™, Invitrogen, Carlsbad Calif.), Sf9 or Sf21,both derived from Spodoptera frugiperda. In various aspects, an insectcell in vitro can comprise a Rep gene of an AAV and/or a Cap gene of anAAV. Hence, in some configurations, an insect cell can comprise a firstnucleic acid cassette and a second nucleic acid cassette, wherein thefirst nucleic acid cassette comprises a Rep 78/68 ORF and a Rep 52/40ORF and the second nucleic acid cassette comprises a VP1 ORF and aVP2/VP3 ORF. These cassettes can each comprise a first insect-operablepromoter and an intron comprising second insect-operable promoter asdescribed herein. Cassettes in some aspects can be comprised within acell by different nucleic acids. In other aspects, cassettes can becomprised by the same nucleic acid, in tandem or anti-senseconfigurations.

In some configurations, an insect cell can further include an additionalnucleic acid comprising a transgene of interest to be expressed by thehost insect cell. Such a nucleic acid can comprise, in some aspects, anadditional cassette comprising, in 5′ to 3′ order, a first invertedterminal repeat (ITR) of an AAV, a mammalian cell-operable promoter; atransgene, a polyadenylation signal, and a second ITR of an AAV. Atransgene of these configurations can comprise an ORF encoding anypolypeptide of interest. In some configurations, a transgene can be areporter gene, such as a chloramphenicol acetyl transferase, aβ-galactosidase, a β-glucoronidase, a renilla luciferase, a fireflyluciferase, a green fluorescent protein (GFP), a red fluorescent protein(RFP) or an alkaline phosphatase such as a secreted alkalinephosphatase. In other configurations, a transgene can comprise an ORFencoding a polypeptide of therapeutic interest, such as, withoutlimitation, a polypeptide hormone, cytokine or growth factor (e.g.,insulin or erythropoietin), an interferon, a blood clotting factor, or avaccine.

In some configurations, a nucleic acid comprising a transgene ofinterest or a cassette comprising a gene having multiple ORFs such as aRep gene and/or a Cap gene of an AAV can be integrated into the genomeof a host insect cell. In some configurations, the integration can be astable integration. Nucleic acids of the present teachings can also beharbored transiently in a host cell.

Some aspects of the present teachings include cell cultures. A cellculture of these aspects comprises a plurality of insect cellscomprising a nucleic acid cassette comprising a first insectcell-operable promoter, a gene having overlapping open reading framesand encodes a plurality of polypeptides, and an intron comprising asecond insect cell-operable promoter, as described supra; and a culturemedium. In some configurations, a cell culture can comprise nucleic acidcassettes which comprise Rep and Cap genes of an AAV. In somearrangements, such cultures can produce AAV, and a culture medium ofthese cultures can have a titer of at least about 10¹³ AAVgenomes/liter, at least about 10¹⁴ AAV genomes/liter, or greater. Insectcells of these configurations can be any insect cell known to skilledartisans, such as cells from cell lines BTI-Tn-5B1-4, Sf9 or Sf21. Cellsof such culture can further comprise a nucleic acid comprising ITRs anda transgene, as described supra.

The present teachings also include methods of expressing a plurality ofpolypeptides encoded by a gene comprising overlapping ORFs. Thesemethods can comprise, in various configurations, infecting, transformingor transfecting at least one insect cell with a nucleic acid cassettecomprising a gene comprising overlapping ORFs as described herein, andculturing the at least one insect cell. Insect cells of theseconfigurations can be any insect cell known to skilled artisans, such ascells from cell lines BTI-Tn-5B1-4, Sf9 or Sf21.

The present teachings also include methods of expressing multiple genesin an insect cell, wherein each gene comprises overlapping ORFs. Thesemethods comprise providing one or more insect cells harboring both afirst nucleic acid cassette comprising a first gene, a firstinsect-operable promoter and an intron comprising a secondinsect-operable promoter as described herein, and a second nucleic acidcassette comprising a second gene, a third insect-operable promoter anda second intron comprising a fourth insect-operable promoter. In variousaspects, the cassettes of these methods can be comprised by the same ordifferent nucleic acids, and the host insect cells can be transiently orstably transformed with either or both cassettes.

Similarly, the present teachings also include methods of producing anadeno-associated virus (AAV) in an insect cell. These methods cancomprise providing one or more insect cells harboring both a firstnucleic acid cassette comprising a Rep gene, a first insect-operablepromoter and an intron comprising a second insect-operable promoter asdescribed herein, and a second nucleic acid cassette comprising a Capgene, which has a third insect-operable promoter and an introncomprising a fourth insect-operable promoter as described herein. Thesemethods further comprise culturing the insect cells in a culture medium.In some configurations, insect cells of these methods can furthercomprise an additional nucleic acid, wherein the additional nucleic acidcomprises a first inverted terminal repeat (ITR) of an AAV; a mammaliancell-operable promoter; a transgene, and a second ITR of an AAV. Invarious configurations, one or more of the cassettes and the additionalnucleic acid can be comprised by one or more vectors.

In some aspects, the present teachings also include methods of producingAAV capsids in insect cells in vitro. These methods comprise: providingone or more insect cells comprising a nucleic acid cassette comprising,in 5′ to 3′ order, a first promoter, a 5′ portion of a Cap gene of anAAV, an intron comprising a second promoter, and a 3′ portion of the Capgene; and culturing the one or more insect cells in a culture medium. Invarious configurations, the nucleic acid can be comprised by a vector.In some configurations, providing insect cells can comprisetransforming, transfecting or infecting one or more insect cells with anucleic acid or vector comprising the cassette. In addition, in someaspects, these methods can further comprise transforming, transfectingor infecting the cells with an additional nucleic acid which comprises afirst inverted terminal repeat (ITR) of an AAV; a mammaliancell-operable promoter; a transgene, and a second ITR of an AAV.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 illustrates a genetic and transcriptional map of representativerecombinant baculovirus expressing both Rep78 or 68 and Rep52 or 40within a single expression cassette. Mature Rep78 or 68 mRNA is formedwhen the artificial intron is removed through splicing. Rep52 or 40 mRNAis transcribed from the promoter located inside the artificial intron.

FIG. 2 illustrates a genetic and transcriptional map of representativerecombinant baculovirus expressing VP1, VP2, and VP3 within a singleexpression cassette. Mature VP1 mRNA is formed when the artificialintron is removed through splicing. RNA encoding VP2 and VP3 istranscribed from the promoter located inside the artificial intron.

FIG. 3 illustrates a genetic map of a three-vector system for parvoviralvector production in insect cells.

FIG. 4 illustrates a genetic map of a two-vector system for parvoviralvector production in insect cells.

FIG. 5 illustrates two examples of a single-vector system together witha stable insect cell line for producing AAV vectors in insect cells.

FIG. 6 illustrates an example of a method for parvoviral genomeproduction.

FIG. 7 illustrates an example of a method for empty parvoviral particleproduction.

FIG. 8 illustrates a genetic map of a recombinant vector comprising AAV2Rep coding sequence comprising an artificial intron comprising apolyhedrin (polh) promoter. This vector can be used for the productionof AAV vectors in insect cells.

FIG. 9 illustrates representative results of Western blots of Rep andCap proteins. (A) AAV2 Rep78 and Rep52 expressed from Sf9 cells infectedwith Bac-inRep; (B) AAV2 Cap proteins expressed from Sf9 cells infectedwith Bac-inCap; (C) AAV8 Cap proteins expressed from Sf9 cells infectedwith Bac-inCap8; (D) AAV6 Cap proteins expressed from Sf9 cells infectedwith Bac-inCap6; and (E) AAV1 Cap proteins expressed from Sf9 cellsinfected with Bac-inCap1.

FIG. 10 illustrates a genetic map of a recombinant vector comprisingAAV2 Cap coding sequence and an artificial intron comprising polyhedrin(polh) promoter. This vector can be used for the production of AAV2vectors in insect cells.

FIG. 11 illustrates a genetic map of a recombinant vector comprisingAAV8 Cap coding sequence and an artificial intron comprising polyhedrin(polh) promoter. This vector can be used for the production ofAAV8-pseudotyped vectors in insect cells.

FIG. 12 illustrates a genetic map of a recombinant vector comprisingAAV6 Cap coding sequence comprising and an artificial intron comprisingpolyhedrin (polh) promoter. This vector can be used for the productionof AAV6-pseudotyped vectors in insect cells.

FIG. 13 illustrates a genetic map of a recombinant vector comprisingAAV1 Cap coding sequence and an artificial intron comprising apolyhedrin (polh) promoter. This vector can be used for the productionof AAV1-pseudotyped vectors in insect cells.

FIG. 14 illustrates a genetic and transcriptional map of recombinantbaculovirus expressing all three SV40 VP proteins within a singleexpression cassette. Mature VP2 mRNA is formed when both the first andsecond introns are removed through splicing and mature VP3 mRNA isformed when the second intron is removed through splicing. The VP1 mRNAis transcribed from the promoter located inside the second intron.

DETAILED DESCRIPTION

The methods and compositions described herein utilize laboratorytechniques well known to skilled artisans, and can be found inlaboratory manuals such as Sambrook, J., et al., Molecular Cloning: ALaboratory Manual, 3rd ed. Cold Spring Harbor Laboratory Press, ColdSpring Harbor, N.Y., 2001; Methods In Molecular Biology, ed. Richard,Humana Press, NJ, 1995; Spector, D. L. et al., Cells: A LaboratoryManual, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y.,1998; and Harlow, E., Using Antibodies: A Laboratory Manual, Cold SpringHarbor Laboratory Press, Cold Spring Harbor, N.Y., 1999. Additionalreferences describing methods of expression of heterologous polypeptidesin insect cells, as well as methods of introducing vectors and nucleicacids into insect cells and methods of maintaining insect cell culturesinclude, for example, O'Reilly et al., Baculovirus Expression Vectors, ALaboratory Manual, Oxford Univ. Press, 1994; Samulski et al., J. Vir.63: 3822-3288, 1989; Kajigaya et al., Proc. Nat'l. Acad. Sci. USA 88:4646-4650, 1991; Ruffing et al., J. Vir. 66: 6922-6930, 1992; Kimbaueret al., Vir. 219: 37-44, 1996; Zhao et al., Vir. 272: 382-393, 2000; andSamulski et al., U.S. Pat. No. 6,204,059.

As used in the description and the appended claims, the singular forms“a”, “an” and “the” are intended to include the plural forms as well,unless the context indicates otherwise. All publications, patentapplications, patents, and other references mentioned herein areincorporated by reference in their entirety.

The present inventor has developed a method for expressing a gene withoverlapping reading frames in insect cells. The inventor has discoveredthat incorporating into a single gene an artificial intron comprising aninsect cell-operable promoter can provide expression of overlapping openreading frames in an insect cell. “Overlapping reading frames” as usedherein, refers to coding sequence from a gene which can be transcribedinto multiple mRNA molecules from multiple transcription start sites; invarious aspects, these mRNA molecules can be translated into multiplepolypeptides. Overlapping reading frames include coding sequences whichcan be transcribed into mRNA molecules having different translationstart sites (start codons) from one gene, with either frame-shiftedreading frames or non-frame-shifted reading frames.

In some aspects, a cassette of the present teachings can comprise a Repgene, a p10 promoter and an intron comprising a polh promoter. Invarious aspects, upon infection of an insect cell by a baculovirusincluding such a cassette, Rep78 or Rep68 pre-mRNA can be transcribedfrom the p10 promoter, and mature mRNA can be formed by splicing out theartificial intron. In addition, the Rep52 or Rep40 mRNA can betranscribed from a polh promoter located inside the artificial intron.As a result, an insect cell can express both Rep78 (or Rep68) and Rep52(or Rep40) from the same Rep coding sequence, while avoiding the use ofseparate Rep78 and Rep52 sequences. In other aspects, any insectcell-operable promoter can be used instead of a p10 or polh promoter.

In some embodiments, the present teachings relate to the expression ofoverlapping Rep and Cap genes of adeno-associated virus in insect cellsby incorporating into the Rep and Cap genes respectively an artificialintron comprising an insect cell-operable promoter. In some aspects,rAAV can be produced in the insect cells by employing the Rep and Capcoding sequences. By insertion of the artificial intron into the Repcoding sequence, both the Rep78 (or Rep68) and Rep52 (or Rep40) can beexpressed from the single Rep coding sequence without the need to usetwo separate Rep coding sequences (FIG. 1). Likewise, by incorporatingthe artificial intron sequence into the Cap coding sequence, all threeCap proteins, VP1, VP2, and VP3, can be expressed from a single Capcoding sequence (FIG. 2). In some configurations, a Cap gene comprisingan artificial intron obviates the need to mutate the VP1 initiationcodon AUG into ACG as described by U.S. Pat. No. 6,723,551 B2.

In some aspects, the present teachings provides a method of producing anAAV in an insect cell. These methods comprise expressing Rep proteinsand Cap proteins by transcribing mRNAs encoding the Rep proteins and Capproteins. In these methods, at least one vector is introduced into aninsect cell. A vector of these aspects comprises one or more nucleicacid molecules comprising a cassette, each cassette comprising, in 5′ to3′ order, a first insect cell-operable promoter, a 5′ portion of a genecomprising a first ORF of the gene comprising multiple ORFs, an introncomprising a second insect cell-operable promoter, and a 3′ portion ofthe gene comprising at least one additional ORF. In variousconfigurations, an insect cell-compatible vector can comprise a firstnucleotide sequence comprising the Rep coding sequence and at least oneartificial intron comprising an insect cell-operable promoter, and asecond nucleotide sequence comprising the Cap coding sequence and atleast one artificial intron. An insect cell into which such vectors areintroduced can then be maintained under conditions such that AAV isproduced. In some configurations, the insect cell can further comprise anucleic acid comprising at least one AAV ITR. This nucleotide acid canalso include a transgene, such as a gene encoding a reporter polypeptideor a polypeptide of therapeutic interest. In various configurations,this nucleic acid can be introduced into the cell by a vector. Thisvector can be distinct from the vector(s) comprising the Rep and/or Capgenes, or, in some configurations, a single vector can comprise thenucleic acid comprising at least one AAV ITR, the Rep gene and the Capgene.

The inventor has determined that AAV Rep and ITR sequences alsoefficiently cross-complement other AAV Rep and ITR sequences in insectcells. Generally, the Cap proteins, which determine the cellulartropicity of the AAV particle, and related Cap protein-encodingsequences are significantly less conserved than Rep proteins and genesamong different AAV serotypes. In view of the ability Rep and ITRsequences to cross-complement corresponding sequences of otherserotypes, pseudotyped AAV particles comprising the capsid proteins of aserotype (e.g., AAV6) and the Rep and/or ITR sequences of another AAVserotype (e.g., AAV2) can readily be generated. As used herein,“pseudotype” refers to the source of Cap protein in an adeno-associatedvirus. See, e.g., Halbert, C. L., et al., J. Virol. 74: 1524-1532, 2000;Halbert, C. L., et al., J. Virol. 75: 6615-6624, 2001. For example, theinventor has produced high titers of rAAV2/6 and rAAV2/8 (i.e.,pseudotyped AAV comprising the ITRs and Rep sequences of AAV2 and VPsequences derived from AAV6 and AAV8, respectively) in Sf9 cells (seeExample 4). In view of the conserved nature of Rep and ITR sequencesamong AAV serotypes, production of a pseudotyped vector comprising theCap genes of a particular AAV serotype in a packaging cell systemindicates that nonpseudotyped vectors of that serotype also can besuccessfully produced in that system. For example, the efficientproduction of rAAV2/6 and rAAV2/8 in Sf9 cells indicates that rAAV6 andrAAV8 also can be efficiently produced in these cells.

In view of the foregoing, it will be understood that sequences from morethan one AAV serotype can be combined for production of AAV in insectcells. For example, a nucleic acid comprising at least one AAV ITRnucleotide sequence can be derived from one serotype, such as AAV2,while other nucleic acids can comprise open reading frames or codingsequences derived from one or more other serotypes, such as, forexample, serotype 3. In various configurations, nucleic acids of any ofAAV serotypes 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, and 11 can provide a Repgene, a Cap gene, and/or an AAV ITR in the present methods.

In some aspects of these methods, an AAV ITR can be an AAV1, AAV2, or anAAV6 ITR; a nucleic acid comprising the Rep ORFs can comprise an AAV1,an AAV2, or an AAV6 Rep gene; and a nucleic acid comprising the Cap ORFscan comprise an AAV1, an AAV2, or an AAV6 Cap gene.

In some aspects, modified AAV sequences also can be used to produce rAAVin insect cells. For example, nucleotide sequences having at least about70%, at least about 75%, at least about 80%, at least about 85%, atleast about 90%, at least about 95% sequence identity to an AAV1, AAV2,AAV3, and/or AAV4 ITR, Rep, or Cap can be used in place of wild-type AAVITR, Rep, or Cap sequences, provided that rAAV particles are produced ininfected cells. Similarly, amino acid sequences having at least about70%, at least about 75%, at least about 80%, at least about 85%, atleast about 90%, at least about 95% sequence identity to an AAV1, AAV2,AAV3, and/or AAV4 polypeptide sequences can be used in place ofwild-type AAV ITR, Rep, or Cap sequences, provided that rAAV particlesare produced in infected cells.

In various aspects, any insect cell known to a skilled artisan which canbe maintained in culture can be used with the present methods.Non-limiting examples of cell lines include Sf9 cells from Spodopterafrugiperda, Sf21 cells from Spodoptera frugiperda, Drosophila celllines, or mosquito cell lines, e.g., cell lines derived from Aedesalbopictus. In some aspects, a cell line can be a Spodoptera frugiperdaSf9 cell line.

Any vector known to a skilled artisan can be employed with the presentteachings provided it is insect cell-compatible. The presence of avector in the insect cell need not be permanent. The vectors can beintroduced by any method known, for example by chemical treatment of thecells, electroporation, or infection. In some aspects, a vector can be abaculovirus, a viral vector, or a plasmid.

If three vectors are used, a first vector can comprise a firstnucleotide sequence comprising a first insect cell-operable promoter, aRep coding sequence and an artificial intron comprising a second insectcell-operable promoter; a second vector can comprise a second nucleotidesequence comprising a third insect cell-operable promoter, Cap codingsequence and an artificial intron comprising a fourth insectcell-operable promoter; and a third nucleotide sequence comprising atleast one AAV ITR nucleotide sequence. In FIG. 3, pA is apolyadenylation signal, polh and p10 are transcriptional promoters forexpression in insect cells, and CMV is a mammalian transcriptionalpromoter for expression of a gene in a mammalian cell. ITR is aninverted terminal repeat of AAV.

In some aspects, AAV can be produced using two vectors in accordancewith the disclosed methods. In these aspects, a first vector cancomprise a nucleotide sequence comprising a first insect cell-operablepromoter which is operably linked to a 5′ portion of a Rep codingsequence, an artificial intron comprising a second insect cell-operablepromoter which is operably linked to a 3′ portion of the Rep codingsequence; a third insect cell-operable promoter operably linked to a 5′portion of a Cap coding sequence, and an artificial intron comprising afourth insect cell-operable promoter which is operably linked to a 3′portion of the Cap coding sequence. A second vector of these aspects cancomprise a nucleotide sequence comprising at least one AAV ITRnucleotide sequence. FIG. 4 is a genetic map of an exemplary two-vectorsystem. In FIG. 4, pA is a polyadenylation signal, polh and p10 aretranscriptional promoters for expression in insect cells, and CMV ismammalian transcriptional promoter for expression of a gene in amammalian cell. ITR is the inverted terminal repeat of AAV.

In various configurations, the sequences comprised by each vector can bein any order relative to each other. For example, in some arrangements,a vector can comprise ITRs and a Cap coding sequence, and the Cap codingsequence can be located on the vector such that, upon replication of theDNA between ITR sequences, the Cap coding sequence can be replicated,while in other arrangements, the Cap coding sequences are notreplicated. In other configurations, Rep coding sequence and the Capcoding sequence can be in any order on a vector.

Methods of introduction of nucleic acid sequences into an insect genomeare well known to skilled artisans, as are methods for selecting andidentifying cells harboring introduced nucleic acids. The incorporationinto the genome can be aided for example, the use of a vector comprisingnucleotide sequences with extensive sequence similarity to one or moreregions of a genome of an insect host cell. The use of genetic elements,such as transposons, provide a method for introducing a nucleotidesequence into a genome. In some aspects of the present methods, atransformed cell can be selected or identified with the aid of a markergene which can be encoded by a nucleic acid sequence added to the cell.The some aspects, incorporation of the nucleic acid sequence into a hostcell genome then can be determined by standard methods well known toskilled artisans such as Southern blots or polymerase chain reaction(PCR) assays.

In some aspects, an ITR can be engineered so that binding sites forreplication polypeptides are situated on both strands of the A regionsand D regions, and are located symmetrically, one on each side of thepalindrome. On a double-stranded circular DNA template (e.g., aplasmid), the Rep78- or Rep68-assisted nucleic acid replication can thenproceed in both directions and a single ITR suffices for AAV replicationof a circular vector. Thus, one ITR nucleotide sequence can be used inthe context of the present teachings. However, two or another evennumber of regular ITRs can be used. In some aspects, two ITR sequencesare used.

In various aspects, a nucleic acid comprising at least one AAV ITR canfurther comprise a nucleic acid sequence encoding at least one “geneproduct of interest” or “transgene” for expression in a mammalian cell,located such that it will be incorporated into an AAV genome replicatedin the insect cell. Any nucleic acid can be incorporated for laterexpression in a mammalian cell transfected with the AAV produced inaccordance with the present teachings. For example, the nucleic acid canencode a protein, express antisense RNA, or small interfering RNA(SiRNA). The protein can be a secretory protein, or a protein which willaffect primarily the cell that is infected with the insect-produced AAV.In some aspects, a product of interest can be Rep78 or Rep68.Accordingly, in these aspects, a nucleotide sequence can comprise twonucleic acid sequences, each encoding one gene product of interest forexpression in a mammalian cell. Each of the two nucleic acid sequencesencoding a product of interest can be arranged such that it will beincorporated into an rAAV genome replicated in an insect cell.

In various configurations, a product of interest can be a gene productwhich can be a polypeptide or an RNA molecule. Non-limiting examples ofa polypeptide of interest include proteins such as an enzyme, a clottingfactor, a peptide hormone or a fusion protein. Other examples ofproducts of interest include a gene product which complements a geneticdefect, an RNA molecule, or a transcription factor. For example, a geneproduct of interest can comprise a nucleotide sequence that provides aregulatory function (e.g., a transposon). Examples of gene products ofinterest include, but are not limited to: hormone receptors (e.g.,mineralcorticosteroid, glucocorticoid, and thyroid hormone receptors);intramembrane proteins (e.g., TM-1 and TM-7); intracellular receptors(e.g., orphans, retinoids, vitamin D3 and vitamin A receptors);signaling molecules (e.g., kinases, transcription factors, and signaltransducers and activators of transcription receptors of the cytokinesuperfamily (e.g. erythropoietin, growth hormone, interferons, andinterleukins, and colony-stimulating factors; G-protein coupledreceptors, e.g., hormones, calcitonin, epinephrine, gastrin, andparacrine or autocrine mediators, such as stomatostatin orprostaglandins; neurotransmitter receptors (norepinephrine, dopamine,serotonin or acetylcholine); pathogenic antigens, which can be of viral,bacterial, allergenic, or cancerous origin; and ligands of tyrosinekinase receptors (such as insulin growth factor, and nerve growthfactor).

In various aspects, a gene product of interest can be a therapeutic geneproduct. A therapeutic gene product can be a polypeptide, RNA molecule,or other gene product that, when expressed in a target cell, provides atherapeutic effect, such as, for example, ablation of an infected cell(e.g., as described by Goldsmith et al., WO 90/07936), expression of apolypeptide having a therapeutic biological activity, and/or expressionof an RNA molecule for antisense therapy (e.g., regulation of expressionof a endogenous or heterologous gene in a target cell genome). Forexample, in a patient about to receive a heterologous transplant orgraft, one may administer a polynucleotide encoding a toxin to T cellstargeting the graft.

An AAV protein can be a gene product of interest. For example, thesequence of a Rep protein, such as Rep78 or Rep68, or a functionalfragment thereof can be a gene product of interest for expression in amammalian cell. A nucleic acid sequence encoding Rep78 and/or Rep68, ifpresent in a rAAV genome of the present teachings and expressed in amammalian cell transduced with the rAAV produced in accordance with thepresent teachings, allows for integration of the rAAV into the genome ofthe transduced mammalian cell. Expression of Rep78 and/or Rep68 in anrAAV-transduced or infected mammalian cell can bestow an advantage forcertain uses of the rAAV produced in an insect cell, such as allowinglong term or permanent expression of any other gene product of interestintroduced in the cell by the rAAV.

A selectable marker is one type of a gene product of interest.Expression of a protein encoded by the selectable marker allows a hostcell transfected with an expression vector which includes the selectablemarker to be distinguished from a host cell which does not have theexpression vector encoding the selectable marker. An example is a hostcell which can use the selectable marker to survive a selection processthat would otherwise kill the host cell, such as treatment with anantibiotic. Such a selectable marker can be one or more antibioticresistance factors, such as neomycin resistance (e.g., neo), hygromycinresistance, and puromycin resistance. A selectable marker also can be acell-surface marker, such as nerve growth factor receptor or truncatedversions thereof. Cells that express the cell-surface marker then can beselected using an antibody targeted to the cell-surface marker. Theantibody targeted to the cell surface marker can be directly labeled(e.g., with a fluorescent substrate) or can be detected using asecondary labeled antibody or substrate which binds to the antibodytargeted to the cell-surface marker. Alternatively, cells can benegatively selected by using an enzyme, such as Herpes simplex virusthymidine kinase (HSVTK) that converts a pro-toxin (gancyclovir) into atoxin or bacterial Cytosine Deaminase (CD) which converts the pro-toxin5′-fluorocytosine (5′-FC) into the toxin 5′-fluorouracil (5′-FU).Alternatively, any nucleic acid sequence encoding a polypeptide can beused as a selectable marker as long as the polypeptide is easilyrecognized by an antibody.

A nucleic acid encoding a selectable marker can encode, for example, abeta-lactamase, a luciferase, a green fluorescent protein (GFP), abeta-galactosidase, or other reporter gene as that term is understood inthe art, including cell-surface markers, such as CD4 or the truncatednerve growth factor (NGFR) (for GFP, see WO 96/23810; Heim et al.,Current Biology 2:178-182 (1996); Heim et al., Proc. Natl. Acad. Sci.USA (1995); or Heim et al., Science 373:663-664 (1995); forbeta-lactamase, see WO 96/30540). In some aspects, a selectable markercan be a beta-lactamase. The nucleic acid encoding a selectable markercan encode, for example, a fluorescent protein. A fluorescent proteincan be detected by determining the amount of any quantitativefluorescent property, e.g., the amount of fluorescence at a particularwavelength, or the integral of fluorescence over an emission spectrum.Techniques for measuring fluorescence are well-known to persons of skillin the art.

In various aspects, a nucleic acid for expression in the mammalian cellcan be incorporated into the AAV genome produced in the insect cell ifit is located between two regular ITRs, or is located on either side ofan ITR engineered with two D regions.

In various aspects, a nucleotide sequence encoding a gene product ofinterest for expression in a mammalian cell can be operably linked to atleast one mammalian cell-compatible expression control sequence, e.g., apromoter. Many such promoters are known in the art. It will beunderstood by a skilled artisan that promoters of these aspects includethose that are inducible, tissue-specific, and/or cell cycle-specific.For example, an E2F promoter can mediate tumor-selective, and, inparticular, neurological cell tumor-selective expression in vivo bybeing de-repressed in such cells in vivo. Parr et al., Nat. Med. 3:1145-1149 (1997). In addition, in some configurations, more than oneexpression control sequence can be operably linked to a given nucleotidesequence. For example, a promoter sequence, a translation initiationsequence, and a stop codon can be operably linked to a nucleotidesequence.

Splice sites are sequences on a mRNA which facilitate the removal ofparts of the mRNA sequences after the transcription (formation) of themRNA. Typically, the splicing occurs in the nucleus, prior to mRNAtransport into a cell's cytoplasm.

In some aspects, an expression control sequence can share sequenceidentity with known expression control sequences. A determination of thedegree of sequence identity of two nucleic acids sequences is adetermination of the percentage of time a nucleotide, from among thefour known natural nucleotides, exactly matches a counterpart on asecond nucleotide sequence, i.e., a T matches a T, an A matches an A, aG matches a G, and a C matches a C. A sequence identity of at least 50%,60%, 70%, 80%, 90% or more, can be considered to have substantialsequence similarity with an expression control sequence. In someaspects, sequence identity can be calculated between sequences withoutintroduction of gaps in one or both of the sequences being compared.

A skilled artisan will understand that in order to optimize the sequencesimilarity between two nucleotide sequences, gaps can be introduced ineither or both of the two sequences. In some aspects, if gaps areintroduced, only nucleotides of a first sequence which pair with anucleotide in a second nucleotide sequence (whether or not there is amatch) are used to calculate percentage homology. Algorithms that haveworked out the rules of calculation of percentage homology are known.Examples of such programs include the SIM, GAP, NAP, LAP2, GAP2, ALIGN,BLAST, and PIPMAKER.

For example, the ALIGN program produces an optimal alignment of twochosen protein or nucleic acid sequences using a modification of thedynamic programming algorithm described by Myers and Miller, CABIOS, 4,11-17 (1988). Preferably, if available, the ALIGN program is used withweighted end-gaps. If gap opening and gap extension penalties areavailable, they are preferably set between about −5 to −15 and 0 to −3,respectively, more preferably about −12 and −0.5 to −2, respectively,for amino acid sequence alignments, and −10 to −20 and −3 to −5,respectively, more preferably about −16 and −4, respectively, fornucleic acid sequence alignments. The ALIGN program and principlesunderlying it are further described in, e.g., Pearson et al., Proc.Natl. Acad. Sci. USA, 85: 2444-48 (1988), and Pearson et al., MethodsEnzymol. 183:63-98 (1990).

The BLAST programs provide analysis of at least two amino acid ornucleotide sequences, either by aligning a selected sequence againstmultiple sequences in a database (e.g., GenSeq), or, with BL2SEQ,between two selected sequences. BLAST programs are preferably modifiedby low complexity filtering programs such as the DUST or SEG programs,which are preferably integrated into the BLAST program operations (see,e.g., Wooton et al., Compu. Chem., 17:149-63 (1993); Altschul et al.,Nat. Genet., 6: 119-29 (1994); Hancock et al., Comput. Appl. Biosci.,10:67-70 (1994); and Wootton et al., Meth. in Enzym., 266:554-71(1996)). If a lambda ratio is used, preferred settings for the ratio arebetween 0.75 and 0.95, more preferably between 0.8 and 0.9. If gapexistence costs (or gap scores) are used, the gap existence costpreferably is set between about −5 and −15, more preferably about −10,and the per residue gap cost preferably is set between about 0 to −5,more preferably between 0 and −3 (e.g., −0.5). Similar gap parameterscan be used with other programs as appropriate. The BLAST programs andprinciples underlying them are further described in, e.g., Altschul etal., J. Mol. Biol., 215: 403-10 (1990), Karlin and Altschul, Proc. Natl.Acad. Sci. USA, 87: 2264-68 (1990) (as modified by Karlin and Altschul,Proc. Natl. Acad. Sci. USA, 90: 5873-77 (1993)), and Altschul et al.,Nucl. Acids Res., 25: 3389-3402 (1997).

In some aspects of the methods disclosed herein, it is possible to useless than the four Rep enzymes, such as only one of the Rep78/Rep68enzymes and only one of the Rep52/Rep40 enzymes, wherein both of theenzymes are expressed from the same single Rep coding sequencecomprising the artificial intron. Since the AAV p5 and p19 promotersfunction poorly in insect cells, an insect cell-operable promoter, e.g.,p10 or polh promoter, replaces the p5 and p19 promoters for Rep78/68 andRep52/40. Because the p19 promoter is located in the Rep coding region,replacing p19 promoter with any other promoter changes the codons of RepORF and therefore the functions of Rep78/68 protein.

Methods of the present teachings utilize an artificial intron thatfunctions in insect cells and provides a method of inserting an insectcell-operable promoter into the p19 promoter area without changing theRep78/68 coding sequence and functions, and makes it possible to expressboth Rep78 and Rep52 or Rep68 and Rep40 from a single expressioncassette. In some aspects, the Rep coding sequence can comprise anartificial intron comprising the polh promoter (FIG. 1). In someconfigurations, the sequence of the artificial intron comprising thepolh promoter can be:GTAAGTACTCCCTATCAGTGATAGAGATCTATCATGGAGATAATTAAAATGATAACCATCTCGCAAATAAATAAGTATTTTACTGTTTTCGTAACAGTTTTGTAATAAAAAAACCTATAAATATTCCGGATTATTCATACCGTCCCACCATCGGGCGCGAAGGGGGAGACCTGTAGTCAGAGCCCCCGGGCAGCACACACTGACATCCA CTCCCTTCCTATTGTTTCAG(SEQ ID NO: 1). The polh promoter within this artificial intron isATCATGGAGATAATTAAAATGATAACCATCTCGCAAATAAATAAGTATTTTACTGTTTTCGTAACAGTTTTGTAATAAAAAAACCTATAAATATTCCGGATTATTCATACCGTCCCACCATCGGGCGCG (SEQ ID NO: 20). In some aspects, theartificial intron can be inserted into the Rep coding sequence betweennucleotides 850 and 851 according to the AAV genome (NCBI accession no.AF043303) such that the Rep52 or Rep40 protein can be expressed from thepolh promoter, while the Rep78 or Rep68 protein can be synthesized fromthe p10 promoter located upstream of the Rep coding sequence. In variousother aspects, the artificial intron can be inserted into locationsother than nucleotides 850 and 851.

In some aspects, the present teachings disclose using an artificialintron to express all three Cap proteins (VP1, VP2, and VP3) from asingle Cap coding nucleotide without mutating the AUG translationinitiation codon of VP1 protein. In some configurations, an artificialintron is inserted between nucleotides 2227 and 2228 according to theAAV genome (AF043303) such that the VP2 and VP3 proteins can besynthesized from a polh promoter situated within the artificial intron,whereas the VP1 protein can be expressed from a polh promoter locatedupstream of the Cap coding sequence (FIG. 2). Upon infection of insectcells by baculovirus carrying the Cap coding sequence comprising theartificial intron, the VP1 pre-mRNA can be transcribed from the promoterupstream of the Cap coding region and mature mRNA can be formed bysplicing out the artificial intron. The mature mRNA can then betranslated into VP1 protein using its original AUG instead of ACGinitiation codon so that the original VP1 amino acid composition is notaltered as described by U.S. Pat. No. 6,723,551 B2. In addition, the VP2and VP3 mRNA can be transcribed from the polh promoter located insidethe artificial intron. In accordance with a preferred embodiment, bothof the promoters located upstream of the Cap coding sequence and insidethe artificial intron are the polh promoter.

In various aspects of the present teachings, variations of theartificial intron sequence can be used in the disclosed methods. In someconfigurations, a sequence with substantial sequence similarity to anartificial intron nucleotide sequence can be utilized. For example, asequence of at least 60%, 70%, or 90% sequence identity to theartificial intron nucleotide sequence of SEQ ID NO: 1 can be introducedinto a cassette such that both the Rep78 (or Rep68) and Rep52 (orRep40), or all three Cap proteins (VP1, VP2, and VP3) can be expressed.

In various aspects of the present teachings, an insect cell-compatiblevector comprising at least one nucleotide sequences of the presentteachings is provided. In some configurations, a vector can comprise anAAV Rep-encoding nucleotide sequence, and further comprise an artificialintron comprising an insect cell-operable promoter. In accordance withanother configuration, an insect cell-compatible vector can comprise anAAV Cap-encoding nucleotide sequence, and further comprise an artificialintron of nucleotide sequence of SEQ ID NO: 1 or a nucleotide sequencehaving substantial sequence identity with the sequence set forth as SEQID NO: 1. In some configurations, the AAV capsid proteins VP1, VP2 andVP3 can be from AAV2. FIG. 5 provides two examples of a single vectortogether with a stable cell line for AAV production.

In various aspects of the present teaching, an insect cell is disclosedcomprising at least one of a first nucleotide sequence, a secondnucleotide sequence and a third nucleotide sequence. In these aspects, afirst nucleotide sequence can comprise a first insect cell-operablepromoter, a 5′ portion of Rep-encoding nucleotide sequence, anartificial intron comprising a second insect cell-operable promoter, anda 3′ portion of the Rep-encoding nucleotide sequence, wherein the firstinsect cell-operable promoter is operably linked to the 5′ portion ofthe Rep-encoding nucleotide sequence and the second insect cell-operablepromoter is operably linked to the 3′ portion of the Rep-encodingnucleotide sequence. Furthermore, in these aspects, a second nucleotidesequence can comprise a third insect cell-operable promoter, a 5′portion of a Cap-encoding nucleotide sequence, an artificial introncomprising a fourth insect cell-operable promoter, and a 3′ portion ofthe Cap-encoding nucleotide sequence, wherein the third promoter isoperably linked to the 5′ portion of the Cap-encoding nucleotidesequence and the fourth promoter is operably linked to the 3′ portion ofthe Cap-encoding nucleotide sequence. In addition, an insect cell ofthese teachings can comprise a first nucleotide sequence comprising atleast one AAV ITR nucleotide sequence.

In some aspects of the present teachings, a nucleotide sequencecomprised by an insect cell can comprise two AAV ITR nucleotidesequences and at least one nucleotide sequence encoding a gene productof interest or a transgene for expression in a mammalian cell betweenthe two AAV ITR nucleotide sequences. In various configurations, atleast one of the first, second, and third nucleotide sequences can bestably integrated in the insect cell.

In another aspect, the present teachings provide a method of producing aparvoviral genome in an insect cell. In the method, as illustrated inFIG. 6, one or two insect cell-compatible vectors can be introduced toan insect cell. These vectors can collectively comprise a firstnucleotide sequence and a second nucleotide sequence. A first nucleotidesequence of these aspects can comprise a first insect cell-operablepromoter, a 5′ portion of a Rep coding sequence, an artificialintron-comprising a second insect cell-operable promoter, and a 3′portion of the Rep coding sequence, wherein the first promoter isoperably linked to the 5′ portion of the Rep coding sequence and thesecond promoter is operably linked to the 3′ portion of the Rep codingsequence. A second nucleotide sequence of these aspects can comprise asecond nucleotide sequence that includes at least one parvoviral ITR. Invarious configurations of the methods, after introducing the vector orvectors to an insect cell, the insect cell can be maintained underconditions such that a parvovirus genome is produced therein. Theparvoviral genome can be any nucleic acid that (1) comprises 5′ and 3′ITRs from or having substantial sequence identity (e.g., at least about70% identity, at least about 80% identity, at least about 90% identity,at least about 95% identity, or greater) to AAV 5′ and 3′ ITRs,respectively, and (2) can replicate in the insect cell upon theintroduction of one or more vectors. In some configurations, theparvoviral genome can further include Rep sequences or sequences sharingsubstantial identity thereof. A parvovirus of these aspects can be anymember of the Parvovirinae. In particular, a parvovirus can be aparvovirus which infects mammals. In some aspects, a parvovirus can be adependovirus such as an AAV, for example a human or a simian AAV. Aparvovirus genome produced in an insect cell in accordance with thepresent teachings can include wild-type and/or modified ITRs, Repsequences, and VP sequences, as well as one or more additionalnucleotide sequences (e.g., one or more transgenes).

In yet another aspect, the present teaching disclose methods ofproducing empty parvoviral particles in an insect cell. In these method,illustrated in FIG. 7, an insect cell-compatible vector can beintroduced to an insect cell, and maintained under conditions such thatan empty parvoviral particle is produced therein. The empty parvoviralparticle can be any parvoviral capsids. The parvovirus can be anysuitable member of the Parvovirinae, such as a parvovirus which infectsmammals. In some aspects, a parvovirus can be a dependovirus, such as ahuman or simian AAV. In various configurations, an empty parvovirusparticle produced in an insect cell can include wild-type and/ormodified Cap sequences.

EXAMPLES

Various aspects of the present teachings can be illustrated by thefollowing non-limiting examples. The following examples areillustrative, and are not intended to limit the scope of the claims. Thedescription of a composition or a method in an example does not implythat a described article or composition has, or has not, been produced,or that a described method has, or has not, been performed, except forresults presented in past tense.

Example 1

This example demonstrates that a single nucleic acid comprising a AAV2Rep coding sequence and an artificial intron comprising the polhpromoter can express both Rep78 and Rep52 proteins.

In these experiments, an artificial intron comprising the polh promoterwas designed using similar splicing donor and acceptor sequences asreported by Chisholm and Henner J Virol. 62(9):3193-3200 (1988). Anartificial intron (SEQ ID NO: 1) is inserted into the Rep78 sequencesuch that the Rep52 mRNA is transcribed from the polh promoter locatedinside the artificial intron, whereas the Rep78 pre-mRNA is transcribedfrom the p10 promoter located upstream from the Rep78 start codon. Uponremoval by splicing of the artificial intron by the host cell, matureRep78 mRNA is formed (FIG. 1). In these experiments, the artificialintron was inserted between nucleotides 850 and 851 according tostandard numbering of the AAV genome (available on the internet athttp://www.ncbi.nlm.nih.gov/entrez/viewer.fcgi?db=nuccore&id=2906016,accession no. AF043303; see also Srivastava, A., et al., J. Virol. 45:555-564, 1983) wherein the sequence of nucleotides 841-860 is agtatttaagcgcctgtttg (SEQ ID NO: 25). The plasmid pAAV-RC (Stratagene, La Jolla,Calif.) was first digested with HindIII and SacI to isolate the backbonefragment (6259 bp). Then the pAAV-RC was digested with DrdI and HindIIIto isolate a 977 bp-fragment. Finally 12 oligos comprising theartificial intron containing the polh promoter were synthesized andannealed. The 6259 bp-backbone fragment, the 977 bp-fragment, and theannealed oligos were ligated to create plasmid pAAV-inRC. The Rep codingsequence containing the artificial intron was amplified by a polymerasechain reaction (PCR) using PCR primers5′-GTGTGTATACCCGCCATGCCGGGGTTTTACGAGAT-3′ (SEQ ID NO:14) and5′-GCGCGCATGCTCCTTCAGAGAGAGTGTCCTCGAGC-3′ (SEQ ID NO:15) and digestedwith restriction endonucleases BstZ17I and SphI, and cloned into theSmaI and SphI sites of pFastBacDual (Invitrogen, Carlsbad, Calif.) tocreate pFBD-inRep (FIG. 8). The 12 oligos used to form the artificialintron are as follows:

(SEQ ID NO: 2) 5′-CAGTGGGCGTGGACTAATATGGAACAGTATTTAAGGTAAGTACTCCCTATCAGTGATAG-3′ (SEQ ID NO: 3)5′-AGATCTATCATGGAGATAATTAAAATGATAACCATCTCGCAAATAAA TAAGTATTTTACT-3′ (SEQID NO: 4) 5′-GTTTTCGTAACAGTTTTGTAATAAAAAAACCTATAAATATTCCGGATTATTCATACCGTC-3′ (SEQ ID NO: 5)5′-CCACCATCGGGCGCGAAGGGGGAGACCTGTAGTCAGAGCCCCCGGGC AGCACACACTGAC-3′ (SEQID NO: 6) 5′-ATCCACTCCCTTCCTATTGTTTCAGCGCCTGTTTGAATCTCACGGAGCGTAAACGGTTGG-3′ (SEQ ID NO: 7) 5′-TGGCGCAGCATCTGACGCAC-3′ (SEQ ID NO:8) 5′-GCGTCAGATGCTGCGCCACCAACCGTTTACGCTCCGTGAGATTCAAA CAG-3′ (SEQ ID NO:9) 5′-GCGCTGAAACAATAGGAAGGGAGTGGATGTCAGTGTGTGCTGCCCGG GGGCTCTGACTAC-3′(SEQ ID NO: 10) 5′-AGGTCTCCCCCTTCGCGCCCGATGGTGGGACGGTATGAATAATCCGGAATATTTATAGGT-3′ (SEQ ID NO: 11)5′-TTTTTTATTACAAAACTGTTACGAAAACAGTAAAATACTTATTTATT TGCGAGATGGTTA-3′ (SEQID NO: 12) 5′-TCATTTTAATTATCTCCATGATAGATCTCTATCACTGATAGGGAGTACTTACCTTAAATA-3′ (SEQ ID NO: 13) 5′-CTGTTCCATATTAGTCCACGCCCACTGGAGCT-3′

The plasmid pFBD-inRep was used to transform DH10Bac competent cells andrecombinant Bacmid DNA containing the Rep coding sequence was isolatedand used to generate recombinant baculovirus Bac-inRep in Sf9 cellsaccording to the manufacturer's protocol (Invitrogen, Carlsbad, Calif.).Sf9 cells were maintained at 28° C. in SF900 II SFM containing 100units/ml of penicillin and 100 μg/ml of streptomycin (Invitrogen,Carlsbad, Calif.).

To express the Rep proteins, Sf9 cells were infected at a multiplicityof infection (m.o.i.) of 1 for 3 days at 28° C. and harvested bycentrifugation at 2,000 rpm for 15 min. The cell pellets were lysed inNuPAGE® LDS Sample Buffer (Invitrogen, Carlsbad, Calif.), boiled for 5min, sonicated for 10 seconds. The lysates were subjected toSDS-polyacrylamide gel electrophoresis (SDS-PAGE). Proteins weretransferred onto PVDF membrane and the Rep78 and Rep52 were detected bymonoclonal antibody clone 303.9 (American Research Products; San Jose,Calif.). The results presented in FIG. 9A show that both Rep78 and Rep52were expressed, indicating that the artificial intron was successfullyspliced out to give rise to a full length mRNA that was translated intoRep78 protein. The results further indicate that the polh promoterlocated inside the intron was functional and full length mRNA coding forRep52 was transcribed and translated into Rep52 protein.

Example 2

This example demonstrates that a single AAV2 Cap coding sequencecomprising the artificial intron comprising the polh promoter canexpress VP1, VP2, and VP3 proteins.

The ORF located at the right side of the wild-type AAV genome codes forthree overlapping capsid proteins, VP1, VP2, and VP3. In mammaliancells, these capsid proteins are synthesized from two spliced mRNAsarising from the p40 promoter. One message is translated into VP1, whileanother transcript encodes VP2 and VP3. The naturally occurringinitiation codon for VP2 is ACG, which is poorly utilized, resulting inribosome scanning through to the VP3 initiation codon (AUG). Thealternate usage of two splice acceptor sites and the poor utilization ofACG initiation codon for VP2 are responsible for the stoichiometry ofVP1, VP2, and VP3 in AAV2-infected mammalian cells and mirrors theprotein ratio in the capsids, 1:1:10. The AAV cap intron is not splicedin insect cells.

In order to express all three capsid proteins from a single codingsequence and preserve the native AUG start codon for VP1 protein, thesame artificial intron as described in Example 1 above was used. It wasinserted in the Cap coding sequence between nucleotides 2227 and 2228according to the AAV genome (AF043303) wherein the sequence ofnucleotides 2221-2230 is cttccagatt (SEQ ID NO: 26). First the plasmidpAAV-RC was digested with BamHI and EcoNI to isolate the backbone 4969bp-fragment. The artificial intron was then amplified from pAAV-inRepusing primers 5′-GCGCGGATCCTGTTAAGATGGCTGCCGATGGTTATCTTCCAGGTAAGTACTCCCTATCAGTGATAGAG-3′ (SEQ ID NO: 16) and5′-ATATCGTCTCGCTGAAACAATAGGAAGGGAGTGGAT-3′ (SEQ ID NO:17). The primerSEQ ID NO: 16 contains a BamHI site and the first 25 nucleotides of VP1coding sequence (ATGGCTGCCGATGGTTATCTTCCAG, SEQ ID NO: 27) and theprimer SEQ ID NO: 17 contains a BsmBI site. The PCR product was thendigested with BamHI and BsmBI. A second PCR fragment was amplified frompAAV-RC using primers 5′-AATTCGTCTCGTCAGATTGGCTCGAGGACACTCTCTCTGA-3′(SEQ ID NO:18) and 5′-TCCCGGAGCCGTCTTAACAG-3′ (SEQ ID NO:19) anddigested with restriction enzymes BsmBI and EcoNI. The backbone fragmentwas ligated with the two PCR fragments to create pAAV-inCap. The entireCap coding sequence comprising the artificial intron was then digestedwith BamHI and SnaBI and ligated to the BamHI and HindIII (blunted byKlenow) sites of pFastBacDual plasmid to create plasmid pFBD-inCap (FIG.10). This plasmid was then used to generate recombinant baculovirusBac-inCap according to the protocol as described in Example 1. Sf9 cellswere infected by Bac-inCap for 3 days at 28° C., harvested and lysed inNuPAGE® LDS Sample Buffer. Proteins were then separated by SDS-PAGE andtransferred to PVDF membrane, and probed with a monoclonal antibodydirected against AAV capsid proteins (clone B1, American ResearchProducts; San Jose, Calif.). The results shown in FIG. 9B indicate thatVP1, VP2, and VP3 proteins were expressed, demonstrating the successfulsplicing of the artificial intron to form a full length VP1 mRNA thatwas translated into VP1 protein. The results further demonstrate thatthe polh promoter located inside the artificial intron was workingproperly to drive the expression of VP2 and VP3 proteins.

Example 3

This example demonstrates that by using the same design of artificialintron, VP1, VP2, and VP3 proteins can be expressed from AAV8, AAV6, andAAV1 serotypes.

The same artificial intron as used in examples supra was inserted intothe Cap coding sequence of AAV serotype 8 between nucleotides 2145 and2146 (accession no. NC_(—)006261) wherein the nucleotide sequence2141-2150 is tccagattgg (SEQ ID NO: 28), AAV serotype 6 betweennucleotides 2232 and 2233 (accession no. NC_(—)001862), whereinnucleotide sequence 2231-2240 is agattggctc (SEQ ID NO: 32), and AAVserotype 1 between nucleotides 2247 and 2248 (accession no.NC_(—)002077), wherein nucleotide sequence 2241-2250 is cttccagatt (SEQID NO: 29, see Example 2). To construct pFBD-inCap8 and pFBD-inCap6, theartificial intron was amplified by PCR from pFBD-inCap using primers5′-ATGCCCTCAGAGAGGTTGTCCTCGAGCCAATCTGAAACAAT-3′ (SEQ ID NO:21) and5′-CCCGGTACCGCATGCTATGC-3′ (SEQ ID NO:22). The amplification product wasdigested with EcoNI and SphI, and the digested product was ligated tothe EcoNI and SphI sites of pFBD-Cap8 and pFBD-Cap6 to createpFBD-inCap8 (FIG. 11) and pFBD-inCap6 (FIG. 12) respectively. To clonethe artificial intron into the Cap coding sequence of AAV1, pFBD-inCap8was digested with EcoNI and XbaI to remove the Cap coding sequence ofAAV8. The Cap coding sequence of AAV1 was PCR amplified from an AAV 1plasmid using primers 5′-GATTGGCTCGAGGACAACCTCTCTG-3′ (SEQ ID NO:23) and5′-GGATCCTCTAGAGTCGACCGCTTACAGGGGACGGGTAAGGT-3′ (SEQ ID NO:24). TheEcoNI and XbaI digested PCR fragment was ligated to the EcoNI and XbaIdigested pFBD-inCap8 to create pFBD-inCap1 (FIG. 13). Recombinantbaculoviruses Bac-inCap8, Bac-inCap6, and Bac-inCap1 were generatedaccording to the manufacturer's protocol as described in example 1. Sf9cells were infected by Bac-inCap8, Bac-inCap6, and Bac-inCap1respectively for 3 days at 28° C., harvested and lysed in NuPAGE® LDSSample Buffer. Proteins were separated by SDS-PAGE and transferred toPVDF membrane. The VP1, VP2, and VP3 proteins were detected bymonoclonal antibody B1 clone (American Research Products; San Jose,Calif.). The results, shown in FIG. 9, indicate that VP1, VP2, and VP3proteins were successfully expressed from all the serotypes tested,demonstrating the successful splicing of the artificial intron to form afull length VP1 mRNA that was translated into VP1 proteins. Theseresults further demonstrate that the polh promoter inside the artificialintron was working properly in all serotypes tested to drive theexpression of VP2 and VP3 proteins.

Example 4

This example demonstrates that rAAV can be produced in insect cells byusing the AAV Rep and Cap coding sequences, each comprising anartificial intron.

In these experiments, Sf9 cells were grown at 28° C. to about 10⁷cells/ml in SF900 II SFM media containing 100 units/ml of penicillin and100 μg/ml streptomycin, and diluted to about 5×10⁶ cells/ml prior toinfection. Triple infection was employed to produce rAAV. A m.o.i. of 1of each Bac-inRep, Bac-GFP (or Bac-RFP), and Bac-inCap was used toinfect the Sf9 cells at 28° C. for 3 days to produce AAV type 2 vectors.For AAV type 1, 6, and 8 vector production, Bac-inCap was simplysubstituted by Bac-inCap1, Bac-inCap6, and Bac-inCap8, respectively inthe triple infection. After 3 days of infection, cell pellets werecollected by centrifugation at 2,000 rpm for 15 min in a tabletopcentrifuge. The cell pellets were lysed in lysis buffer as described byUrabe et al., Hum Gene Ther. 1; 13(16):1935-43 (2002) and cellularnucleic acids (DNA and RNA) were digested by benzonase (Sigma, St.Louis, Mo.). The cell lysates were cleared by centrifugation at 8,000rpm for 30 min in an Avanti J-25 centrifuge (Backman, Fullerton, Calif.)and then loaded onto an SW28 centrifuge tube containing 5 ml of 1.55g/cc, and 10 ml of 1.32 g/cc of CsCl solutions. After centrifugation at28,000 rpm for about 16 hours at 15° C., the rAAV-containing fractionwas collected by puncturing the centrifuge tube using a syringe needleand subjected to a second round of CsCl ultracentrifugation. TherAAV-containing fraction was collected again by puncturing thecentrifuge tube using a syringe needle and dialyzed in PBS buffer toremove the salts and detergents. Vector titers were determined byquantitative real-time PCR assay according to manufacturer's protocol(Applied Biosystems, Foster City, Calif.). The results, presented inTable 1, show that high titers of rAAV vectors can be produced in Sf9cells using the recombinant baculoviruses that carry the Rep and Capcoding sequences comprising the artificial intron, respectively.

TABLE 1 AAV vector genome yields in Sf9 cells as determined byquantitative RT-PCR Experiment Yield (vector genome/liter of Sf9 No.Serotype & Transgene culture) 1 AAV2-GFP 1.56 × 10¹⁴ 2 AAV2-RFP 1.58 ×10¹⁴ 3 AAV2-GFP 9.77 × 10¹³ 4 AAV6-GFP 3.53 × 10¹³ 5 AAV8-GFP 9.65 ×10¹³ 6 AAV1-GFP 4.36 × 10¹³ 7 AAV1-GFP 4.47 × 10¹³

Example 5

This example demonstrates the production of rAAV in insect cells usingthe two-vector system.

The Rep and Cap coding sequence each comprising the artificial intronwere cloned together using standard cloning techniques into a singlebaculovirus as shown in FIG. 4. Sf9 cells were grown at 28° C. to 10⁷cells/ml and diluted to 5×10⁶ cells/ml right before infection.Bac-inRep-inCap and Bac-GFP each at m.o.i. of one were used to infectthe cells at 28° C. for 3 days. The cells were harvested and rAAVvectors were purified as described in Example 4. The results, presentedin TABLE 2, indicate the successful production of AAV2 vectors using thetwo-vector system.

TABLE 2 AAV vector genome yields in Sf9 cells as determined byquantitative RT-PCR Experiment Yield (vector genome/liter of Sf9 No.Serotype & Transgene culture) 1 AAV2-GFP 1.33 × 10¹⁴ 2 AAV2-GFP 1.06 ×10¹⁴

Example 6

This example demonstrates that VP1, VP2, and VP3 proteins are properlypackaged in virions by using AAV Cap coding sequences comprising theartificial intron.

In these experiments, purified AAV2, AAV6, and AAV8 vectors, each at10¹⁰ vector genomes, were boiled in NuPAGE® LDS Sample Buffer for 5 min.Proteins were resolved by SDS-PAGE and transferred to PVDF membrane. TheVP1, VP2, and VP3 proteins were detected by monoclonal antibody B1 cloneas described in Example 2. The results demonstrate that by using the Capcoding sequences comprising the artificial intron, all three capsidproteins can be properly packaged into virions.

Example 7

This example illustrates that AAV vectors produced in the insect cellsusing the Rep and Cap coding sequences comprising the artificial intronare infectious and can deliver genes to target cells.

In these experiments, AAV2-GFP and AAV6-GFP were used to transduce HepG2hepatocellular carcinoma cells (American Type Culture Collection,Manassas, Va.) to show the infectivity of the vectors produced in insectcells. HepG2 cells were grown at 37° C. in MEM medium (ATCC)supplemented with 10% fetal bovine serum (Hyclone, Logan, Utah) and 100units/ml of penicillin and 100 μg/ml of streptomycin (Invitrogen,Carlsbad, Calif.). HepG2 cells at 1.5×10⁵ cells/well were seeded in24-well plate and grown overnight at 37° C. in a CO₂ incubator. AAVvectors were diluted to 1.2×10⁹ vg/ml, 1.2×10⁸ vg/ml, 1.2×10⁷ vg/ml, and1.2×10⁶ vg/ml in the culture medium without serum but containing 20 μMof etoposide (A.G. Scientific, Inc., San Diego, Calif.). Old media wereremoved from the cells and 500 μl of diluted AAV vectors were added toeach well. Two days after transduction, GFP expressing cells were scoredand photographed. The results show that HepG2 cells were efficientlytransduced both by AAV2-GFP and AAV6-GFP vectors.

Example 8

This example illustrates that nucleic acid sequences of the presentteachings comprising Rep- or Cap-encoding sequences and an artificialintron are stable in a baculovirus.

To demonstrate the stability of baculoviruses comprising the Rep and Capcoding sequences respectively, the baculoviruses were plaque purifiedand subsequently passaged for 5 times, and Rep and Cap proteinexpression was assayed. The plaque purification was performed asfollows: Sf9 cells at 1×10⁶ cells/well were seeded in 6-well plate andincubated at 28° C. for 30 min. The baculoviruses were diluted to 100,50, and 25 pfu/100 μl. Old media were removed from the cells and thediluted baculoviruses were added to infect the cells for 20 min at 28°C. Agarose in DPBS at 1% was melted, cool to 37° C., and mixed with 1volume of SF900II SFM at 37° C., and 1.5 ml of the agarose-SF900II SFMoverlay was added to each well. When the agarose solidified, 1.5 ml ofSF900II SFM was added to each well and the plates were incubated at 28°C. for 6 days to let plaques form. By the end of incubation, 6 plaquesfrom Bac-inCap and 12 plaques from Bac-inRep were picked and transferredto microfuge tubes containing 500 μl SF900II SFM media. Sf9 cells in6-well plates were infected with 100 μl of each plaque for 4 days. Thecells were collected for Western blot analysis and the supernatants werecollected and 3 μl of the supernatants were used to infect Sf9 cells in6-well plates. This procedure was repeated for 4 more times untilpassage 5. The results show that all the plaques picked through 5passages express the Rep78 and Rep52 or the VP1, VP2, and VP3 proteinsas expected, with no apparent loss of protein expression.

Example 9

This example demonstrates expression in an insect cell of multiple SV40VP proteins from a single expression cassette comprising multipleintrons.

Simian virus 40 (SV40) is a double-stranded DNA virus with a covalentlyclosed circular genome of 5.2 kb, and has been sequenced in its entirety(Fiers, W., et al., Nature 273: 113-120, 1978). The sequence isavailable on the internet athttp://www.ncbi.nlm.nih.gov/entrez/viewer.fcgi?db=nuccore&id=9628421,accession no. NC_(—)001669. In mammalian cells, its three capsidproteins, VP1, VP2, and VP3 are all transcribed from the same SV40promoter and expression of these VP proteins is controlled by mechanismof intron splicing. The VP3 protein is a truncated form of the VP2protein and the 5′ portion the sequence encoding the VP1 protein isoverlapping with the 3′ portion of VP2 and VP3 but does not share thesame ORF.

Artificial introns can be used to express SV40 capsid proteins in insectcells. To drive VP3 expression, an artificial intron comprising a polhpromoter is inserted into the SV40 genome between nucleotides 913 and914 wherein the sequence of nucleotides 911-920 is caggaatggc (SEQ IDNO: 30). To drive VP1 expression, an artificial intron comprising thesame polh promoter is inserted between nucleotides 1462 and 1463 whereinthe sequence of nucleotides 1461-1470 is aggcctgtac (SEQ ID NO: 31). TheVP2 protein is expressed from the polh promoter operably linked to theVP2 gene (FIG. 14).

All publications and patent applications cited in this specification areherein incorporated by reference as if each individual publication orpatent application were specifically and individually indicated to beincorporated by reference. Although the foregoing teachings have beendescribed in some detail by way of illustration and example for purposesof clarity of understanding, it will be readily apparent to those ofordinary skill in the art in light of the teachings that certain changesand modifications may be made thereto without departing from the spiritor scope of the appended claims.

REFERENCES

-   1. Berns, K. I., “Parvoviridae: The Viruses and Their Replication,”    Chapter 69 in Fields Virology (3d Ed. 1996).-   2. Choi et al., Curr. Gene Ther., June; 5(3):299-310 (2005).-   3. Tratschin et al., Mol. Cell Biol., 5(11):3251-3260 (1985).-   4. Grimm et al., Hum. Gene Ther., 10(15):2445-2450 (1999).-   5. Jennings et al., Arthritis Res, 3:1 (2001).-   6. Davidson et al., Proc. Natl. Acad. Sci. USA, 97(7):3428-3432    (2000).-   7. Urabi et al., J Virol., 80(4):1874-85 (2006).-   8. Kohlbrenner et al., Mol. Ther., 12(6):1217-25 (2005).-   9. Chao et al., Mol. Ther. 2:619 (2000).-   10. Chiorini et al., J. Virol. 73:1309 (1999).-   11. Xiao et al., J. Virol. 73:3994 (1999).-   12. Muramatsu et al., Virology 221:208 (1996).-   13. Chiorini et al., J. Vir. 71: 6823-33 (1997).-   14. Srivastava et al., J. Vir. 45:555-64 (1983).-   15. Chiorini et al., J. Vir. 73:1309-1319 (1999).-   16. Rutledge et al., J. Vir. 72:309-319 (1998).-   17. Wu et al., J. Vir. 74: 8635-47 (2000).-   18. Samulski et al., J. Vir. 63:3822-8 (1989).-   19. Kajigaya et al., Proc. Nat'l. Acad. Sci. USA 88: 4646-50 (1991).-   20. Ruffing et al., J. Vir. 66:6922-30 (1992)-   21. Kimbauer et al., Vir. 219:37-44 (1996).-   22. Zhao et al., Vir. 272:382-93 (2000).-   23. Chisholm and Henner, J. Virol., 62(9):3193-3200 (1988).

U.S. PATENT DOCUMENTS

5,387,484 A February 1995 Doany 5,688,676 A November 1997 Zhou et al.5,691,176 A November 1997 Lebkowski et al. 5,741,683 A April 1998Russell et al. 6,204,059 B1 March 2001 Samulski et al. 2002/0081721 A1June 2002 Allen et al. 6,723,551 B2 April 2004 Kotin, et al 2006/0166363A1 July 2006 Zolotukhin et al.

What is claimed is:
 1. A nucleic acid cassette for expressing in aninsect cell a plurality of polypeptides encoded by a parvovirus capsidgene, the cassette comprising in 5′ to 3′ order: (i) a first insect celloperable promoter linked operably to a 5′ portion of a first ORF of theparvovirus capsid gene, the first ORF comprising a translationinitiation codon, (ii) an intron comprising a second insect celloperable promoter, the second promoter operably linked to a 5′ portionof an at least one additional ORF of a parvovirus capsid gene, whereinthe at least one additional ORF comprises at least one additionaltranslation initiation codon and overlaps with the 3′ portion of thefirst ORF.
 2. A nucleic acid cassette in accordance with claim 1,wherein the first ORF is a VP1 ORF and the at least one additional ORFis a VP2/VP3 ORF.
 3. A nucleic acid cassette for expressing in an insectcell a plurality of polypeptides encoded by a parvovirus Rep gene, thecassette comprising in 5′ to 3′ order: (i) a first insect cell operablepromoter linked operably to a 5′ portion of a first ORF of theparvovirus Rep gene, the first ORF comprising a translation initiationcodon, (ii) an intron comprising a second insect cell operable promoter,the second promoter operably linked to a 5′ portion of an at least oneadditional ORF of a parvovirus Rep gene, wherein the at least oneadditional ORF comprises at least one additional translation initiationcodon and overlaps with the 3′ portion of the first ORF.
 4. A nucleicacid cassette for expressing in an insect cell a plurality ofpolypeptides encoded by a polyomavirus gene, the cassette comprising in5′ to 3′ order: (i) a first insect cell operable promoter linkedoperably to a 5′ portion of a first ORF of a polyomavirus capsid gene,the first ORF comprising a translation initiation codon, (ii) an introncomprising a second insect cell operable promoter, the second promoteroperably linked to a 5′ portion of an at least one additional ORF of apolyomavirus capsid gene, wherein the at least one additional ORFcomprises at least one additional translation initiation codon andoverlaps with the 3′ portion of the first ORF.
 5. A nucleic acidcassette in accordance with claim 1, 3 or 4, further comprising apolyadenylation signal situated 3′ to the additional ORF in (ii).
 6. Anucleic acid cassette in accordance with claim 1, 3 or 4, wherein eachof the first insect cell-operable promoter and the second insectcell-operable promoter is independently selected from the groupconsisting of a p10 promoter and a polh promoter.
 7. A nucleic acidcassette in accordance with claim 3, wherein the first ORF is a Rep78/68 ORF and the at least one additional ORF is a Rep 52/40 ORF.
 8. Anucleic acid cassette in accordance with claim 1, 3 or 4, wherein thefirst promoter is a p10 promoter and the second promoter is a polhpromoter.
 9. A nucleic acid cassette in accordance with claim 1, 3 or 4,wherein the first promoter is a first polh promoter and the secondpromoter is a second polh promoter.
 10. A vector comprising the nucleicacid cassette of claim 1, 3 or
 4. 11. A vector in accordance with claim10, wherein the vector is selected from the group consisting of aplasmid, a virus and a combination thereof.
 12. An isolated insect cellcomprising the nucleic acid cassette of claim 1, 3 or
 4. 13. An isolatedinsect cell comprising a first nucleic acid cassette of claim 1 and asecond nucleic acid cassette, wherein the first nucleic acid cassettecomprises a Cap gene comprising a VP1 ORF and a VP2/VP3 ORF and thesecond nucleic acid cassette comprises: (i) a first insect cell operablepromoter linked operably to a 5′ portion of a first ORF of theparvovirus Rep gene, the first ORF comprising a translation initiationcodon, (ii) an intron comprising a second insect cell operable promoter,the second promoter operably linked to a 5′ portion of an at least oneadditional ORF of a parvovirus Rep gene, wherein the at least oneadditional ORF comprises at least one additional translation initiationcodon and overlaps with the 3′ portion of the first ORF.
 14. An isolatedinsect cell in accordance with claim 13, further comprising anadditional nucleic acid cassette, the additional cassette comprising, in5′ to 3′ order: a first inverted terminal repeat (ITR) of anadeno-associated virus; a mammalian cell-operable promoter; a transgene;a polyadenylation signal; and a second ITR of an AAV.
 15. An insect cellin accordance with claim 14, wherein the transgene is a reporter geneencoding a polypeptide selected from the group consisting of achloramphenicol acetyl transferase, a β-galactosidase, aβ-glucoronidase, a renilla luciferase, a firefly luciferase, a greenfluorescent protein (GFP), a red fluorescent protein (RFP) and analkaline phosphatase.
 16. An insect cell in accordance with claim 14,wherein the transgene comprises an ORF encoding a polypeptide selectedfrom the group consisting of a polypeptide hormone, an interferon, ablood clotting factor, a vaccine and an erythropoietin.
 17. An insectcell in accordance with claim 12, wherein the nucleic acid cassette isintegrated into the genome of the insect cell.
 18. An insect cell inaccordance with claim 14, wherein at least one of the nucleic acidcassette and the additional nucleic acid cassette is integrated into thegenome of the insect cell.
 19. An insect cell in accordance with claim12, wherein the cell is selected from the group consisting of aTrichoplusia ni BTI-Tn-5B1-4 cell, a Spodoptera frugiperda Sf9 cell anda Spodoptera frugiperda Sf21 cell.
 20. A cell culture comprising: aplurality of insect cells of claim 12; and a culture medium.
 21. A cellculture comprising: a plurality of insect cells of claim 14; and aculture medium comprising at least 10¹³ AAV genomes/liter.
 22. A cellculture in accordance with claim 21, wherein the culture mediumcomprising at least 10¹³ AAV genomes/liter is a culture mediumcomprising at least 10¹⁴ AAV genomes/liter.
 23. A nucleic acidcomprising the cassette of claim 5, further comprising a secondcassette, the second cassette comprising, in 5′ to 3′ order: a secondpolyadenylation signal; a 3′ portion of a second gene comprisingoverlapping ORFs; a second intron comprising a third insect-operablepromoter; a 5′ portion of the second gene; and a fourth insect operablepromoter.
 24. A nucleic acid in accordance with claim 23, wherein thesecond cassette is in an anti-sense orientation relative to the firstcassette.
 25. A nucleic acid in accordance with claim 23, wherein thesecond cassette is in a sense orientation relative to the firstcassette.
 26. A nucleic acid in accordance with claim 23, wherein thefirst cassette comprises an AAV Rep gene comprising a Rep 78/68 ORF anda Rep 52/40 ORF and the second cassette comprises an AAV Cap genecomprising a VP1 ORF and a VP2/VP3 ORF.